Diagnosing fetal chromosomal aneuploidy using massively parallel genomic sequencing

ABSTRACT

Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. The determination of the relative amounts may count sequences of only certain length. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists. Prior to sequencing, the biological sample may be enriched for DNA fragments of a particular sizes.

CLAIM OF PRIORITY

This application claims priority to and is a continuation of U.S.application Ser. No. 12/614,350 filed Nov. 6, 2009 (Attorney Docket No.016285-005221US) which is a continuation-in-part application of U.S.application Ser. No. 12/178,181, entitled “DIAGNOSING FETAL CHROMOSOMALANEUPLOIDY USING MASSIVELY PARALLEL GENOMIC SEQUENCING” filed Jul. 23,2008 (Attorney Docket No. 016285-005220US), which claims priority fromU.S. Provisional Application No. 60/951,438, entitled “DETERMINING ANUCLEIC ACID SEQUENCE IMBALANCE” filed Jul. 23, 2007 (Attorney DocketNo. 016285-005200US), the entire contents of each of the above areherein incorporated by reference for all purposes.

FIELD OF THE INVENTION

This invention generally relates to the diagnostic testing of fetalchromosomal aneuploidy by determining imbalances between differentnucleic acid sequences, and more particularly to the identification oftrisomy 21 (Down syndrome) and other chromosomal aneuploidies viatesting a maternal sample (e.g. blood).

BACKGROUND

Fetal chromosomal aneuploidy results from the presence of abnormaldose(s) of a chromosome or chromosomal region. The abnormal dose(s) canbe abnormally high, e.g. the presence of an extra chromosome 21 orchromosomal region in trisomy 21; or abnormally low, e.g. the absence ofa copy of chromosome X in Turner syndrome.

Conventional prenatal diagnostic methods of a fetal chromosomalaneuploidy, e.g., trisomy 21, involve the sampling of fetal materials byinvasive procedures such as amniocentesis or chorionic villus sampling,which pose a finite risk of fetal loss. Non-invasive procedures, such asscreening by ultrasonography and biochemical markers, have been used torisk-stratify pregnant women prior to definitive invasive diagnosticprocedures. However, these screening methods typically measureepiphenomena that are associated with the chromosomal aneuploidy, e.g.,trisomy 21, instead of the core chromosomal abnormality, and thus havesuboptimal diagnostic accuracy and other disadvantages, such as beinghighly influenced by gestational age.

The discovery of circulating cell-free fetal DNA in maternal plasma in1997 offered new possibilities for noninvasive prenatal diagnosis (Lo,YMD and Chiu, RWK 2007 Nat Rev Genet 8, 71-77). While this method hasbeen readily applied to the prenatal diagnosis of sex-linked (Costa, J Met al. 2002 N Engl J Med 346, 1502) and certain single gene disorders(Lo, YMD et al. 1998 N Engl J Med 339, 1734-1738), its application tothe prenatal detection of fetal chromosomal aneuploidies has representeda considerable challenge (Lo, YMD and Chiu, RWK 2007, supra). First,fetal nucleic acids co-exist in maternal plasma with a high backgroundof nucleic acids of maternal origin that can often interfere with theanalysis of fetal nucleic acids (Lo, YMD et al. 1998 Am J Hum Genet 62,768-775). Second, fetal nucleic acids circulate in maternal plasmapredominantly in a cell-free form, making it difficult to derive dosageinformation of genes or chromosomes within the fetal genome.

Significant developments overcoming these challenges have recently beenmade (Benachi, A & Costa, J M 2007 Lancet 369, 440-442). One approachdetects fetal-specific nucleic acids in the maternal plasma, thusovercoming the problem of maternal background interference (Lo, YMD andChiu, RWK 2007, supra). Dosage of chromosome 21 was inferred from theratios of polymorphic alleles in the placenta-derived DNA/RNA molecules.However, this method is less accurate when samples contain lower amountof the targeted nucleic acid and can only be applied to fetuses who areheterozygous for the targeted polymorphisms, which is only a subset ofthe population if one polymorphism is used.

Dhallan et al (Dhallan, R, et al. 2007, supra Dhallan, R, et al. 2007Lancet 369, 474-481) described an alternative strategy of enriching theproportion of circulating fetal DNA by adding formaldehyde to maternalplasma. The proportion of chromosome 21 sequences contributed by thefetus in maternal plasma was determined by assessing the ratio ofpaternally-inherited fetal-specific alleles to non-fetal-specificalleles for single nucleotide polymorphisms (SNPs) on chromosome 21. SNPratios were similarly computed for a reference chromosome. An imbalanceof fetal chromosome 21 was then inferred by detecting a statisticallysignificant difference between the SNP ratios for chromosome 21 andthose of the reference chromosome, where significant is defined using afixed p-value of <0.05. To ensure high population coverage, more than500 SNPs were targeted per chromosome. However, there have beencontroversies regarding the effectiveness of formaldehyde to enrichfetal DNA to a high proportion (Chung, GTY, et al. 2005 Clin Chem 51,655-658), and thus the reproducibility of the method needs to be furtherevaluated. Also, as each fetus and mother would be informative for adifferent number of SNPs for each chromosome, the power of thestatistical test for SNP ratio comparison would be variable from case tocase (Lo, YMD & Chiu, RWK. 2007 Lancet 369, 1997). Furthermore, sincethese approaches depend on the detection of genetic polymorphisms, theyare limited to fetuses heterozygous for these polymorphisms.

Using polymerase chain reaction (PCR) and DNA quantification of achromosome 21 locus and a reference locus in amniocyte cultures obtainedfrom trisomy 21 and euploid fetuses, Zimmermann et al (2002 Clin Chem48, 362-363) were able to distinguish the two groups of fetuses based onthe 1.5-fold increase in chromosome 21 DNA sequences in the former.Since a 2-fold difference in DNA template concentration constitutes adifference of only one threshold cycle (Ct), the discrimination of a1.5-fold difference has been the limit of conventional real-time PCR. Toachieve finer degrees of quantitative discrimination, alternativestrategies are needed.

Digital PCR has been developed for the detection of allelic ratioskewing in nucleic acid samples (Chang, H W et al. 2002 J Natl CancerInst 94, 1697-1703). Digital PCR is an amplification based nucleic acidanalysis technique which requires the distribution of a specimencontaining nucleic acids into a multitude of discrete samples where eachsample containing on average not more than about one target sequence persample. Specific nucleic acid targets are amplified withsequence-specific primers to generate specific amplicons by digital PCR.The nucleic acid loci to be targeted and the species of or panel ofsequence-specific primers to be included in the reactions are determinedor selected prior to nucleic acid analysis.

Clinically, it has been shown to be useful for the detection of loss ofheterozygosity (LOH) in tumor DNA samples (Zhou, W. et al. 2002 Lancet359, 219-225). For the analysis of digital PCR results, sequentialprobability ratio testing (SPRT) has been adopted by previous studies toclassify the experimental results as being suggestive of the presence ofLOH in a sample or not (El Karoui at al. 2006 Stat Med 25, 3124-3133).

In methods used in the previous studies, the amount of data collectedfrom the digital PCR is quite low. Thus, the accuracy can be compromiseddue to the small number of data points and typical statisticalfluctuations.

It is therefore desirable that noninvasive tests have high sensitivityand specificity to minimize false negatives and false positives,respectively. However, fetal DNA is present in low absoluteconcentration and represent a minor portion of all DNA sequences inmaternal plasma and serum. It is therefore also desirable to havemethods that allow the noninvasive detection of fetal chromosomalaneuploidy by maximizing the amount of genetic information that could beinferred from the limited amount of fetal nucleic acids which exist as aminor population in a biological sample containing maternal backgroundnucleic acids.

BRIEF SUMMARY

Embodiments of this invention provide methods, systems, and apparatusfor determining whether a nucleic acid sequence imbalance (e.g.,chromosome imbalance) exists within a biological sample obtained from apregnant female. This determination may be done by using a parameter ofan amount of a clinically-relevant chromosomal region in relation toother non-clinically-relevant chromosomal regions (background regions)within a biological sample. In one aspect, an amount of chromosomes isdetermined from a sequencing of nucleic acid molecules in a maternalsample, such as urine, plasma, serum, and other suitable biologicalsamples. Nucleic acid molecules of the biological sample are sequenced,such that a fraction of the genome is sequenced. One or more cutoffvalues are chosen for determining whether a change compared to areference quantity exists (i.e. an imbalance), for example, with regardsto the ratio of amounts of two chromosomal regions (or sets of regions).The sensitivity of the change compared to a reference quantity isincreased by enriching the amount of fetal sequences being countedrelative to the amount of maternal sequences being counted. Thus,changes compared to the reference quantity can be more pronounced andaccurate. In one aspect, such enrichment is balanced with a need to havea sufficient number of sequences with which to perform the analysis.

According to one exemplary embodiment, a biological sample received froma pregnant female is analyzed to perform a prenatal diagnosis of a fetalchromosomal aneuploidy. The biological sample includes nucleic acidmolecules. A portion of the nucleic acid molecules contained in thebiological sample are sequenced. Both ends of respective nucleic acidsare sequenced in order to provide a length of each sequence. Forexample, a comparison of both ends to a reference sequence (e.g. theentire genome) may be used to provide the length.

Based on the sequencing, a first amount of a first chromosome isdetermined from sequences identified as originating from the firstchromosome. A second amount of one or more second chromosomes isdetermined from sequences identified as originating from one of thesecond chromosomes. The counting of the first and second amounts isdependent on the lengths of the sequences that are counted. A higherrelative count of sequences from the fetus can thus be provided sincethe proportion of sequences that are maternal relative to fetal is alsolength dependent.

Further, a parameter from the first amount and the second amount is thencompared to one or more cutoff values. Based on the comparison, aclassification of whether a fetal chromosomal aneuploidy exists for thefirst chromosome is determined.

According to another exemplary embodiment, a biological sample receivedfrom a pregnant female is analyzed to perform a prenatal diagnosis of afetal chromosomal aneuploidy. The biological sample includes nucleicacid molecules and has been enriched for sequences less than a firstpredetermined number of nucleotides, where the first predeterminednumber is between about 125 and about 175 nucleotides.

Based on the sequencing, a first amount of a first chromosome isdetermined from sequences identified as originating from the firstchromosome. A second amount of one or more second chromosomes isdetermined from sequences identified as originating from one of thesecond chromosomes. A parameter from the first amount and the secondamount is then compared to one or more cutoff values. Based on thecomparison, a classification of whether a fetal chromosomal aneuploidyexists for the first chromosome is determined. The enrichmentadvantageously provides a higher relative count of sequences from thefetus to provide greater ease in determining an aneuploidy while stillmaintaining a sufficient number of total sequences to prevent errors,such as false positives.

Other embodiments of the invention are directed to systems and computerreadable media associated with methods described herein.

A better understanding of the nature and advantages of the presentinvention may be gained with reference to the following detaileddescription and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a flowchart of a method 100 for performing prenatal diagnosisof a fetal chromosomal aneuploidy in a biological sample obtained from apregnant female subject according to an embodiment of the presentinvention.

FIG. 1B is a flowchart of a method 101 for performing prenatal diagnosisof a fetal chromosomal aneuploidy in a biological sample obtained from apregnant female subject according to an embodiment of the presentinvention.

FIG. 2 is a flowchart of a method 200 for performing prenatal diagnosisof a fetal chromosomal aneuploidy using random sequencing according toan embodiment of the present invention.

FIG. 3A shows a plot of percentage representation of chromosome 21sequences in maternal plasma samples involving trisomy 21 or euploidfetuses according to an embodiment of the present invention.

FIG. 3B shows a correlation between maternal plasma fractional fetal DNAconcentrations determined by massively parallel sequencing andmicrofluidics digital PCR according to an embodiment of the presentinvention.

FIG. 4A shows a plot of percentage representation of aligned sequencesper chromosome according to an embodiment of the present invention.

FIG. 4B shows a plot of difference (%) in percentage representation perchromosome between the trisomy 21 case and euploid case shown in FIG.4A.

FIG. 5 shows a correlation between degree of over-representation inchromosome 21 sequences and the fractional fetal DNA concentrations inmaternal plasma involving trisomy 21 fetuses according to an embodimentof the present invention.

FIG. 6 shows a table of a portion of human genome that was analyzedaccording to an embodiment of the present invention. T21 denote a sampleobtained from a pregnancy involving a trisomy 21 fetus.

FIGS. 7A and 7B show a table of a number of sequences required todifferentiate euploid from trisomy 21 fetuses according to an embodimentof the present invention.

FIG. 8A shows a table of top ten starting positions of sequenced tagsaligned to chromosome 21 according to an embodiment of the presentinvention.

FIG. 8B shows a table of top ten starting positions of sequenced tagsaligned to chromosome 22 according to an embodiment of the presentinvention.

FIG. 9 shows a block diagram of an exemplary computer apparatus usablewith system and methods according to embodiments of the presentinvention.

FIGS. 10 a and 10 b shows a schematic comparison between locus-specificand locus-independent methods for DNA quantification.

FIG. 11 shows plots of z-scores for each chromosome for maternal plasmasamples from 14 trisomy 21 and 14 euploid pregnancies according to anembodiment of the present invention.

FIG. 12 shows a bar chart of proportion of accepted PE reads for eachhuman chromosome for three maternal plasma samples collected in thethird trimester according to an embodiment of the present invention.

FIG. 13 shows a table of a summary of clinical information and sequencecounts for the first and second trimester pregnancies studied accordingto an embodiment of the present invention.

FIG. 14 is a bar chart of the proportion of accepted PE reads forchromosomes 21, X and Y for 13 early pregnancy maternal plasma samplesaccording to an embodiment of the present invention.

FIGS. 15A and 15B show representative results for the size distributionof nucleotide fragments for one adult male plasma sample and onematernal plasma, respectively.

FIG. 16 is a plot showing the proportions of retained reads at aplurality of size cutoffs according to an embodiment of the presentinvention.

FIG. 17 shows the amount of retained reads from chrY as a proportion ofall retained reads, termed retained % chrY according to an embodiment ofthe present invention.

FIG. 18A shows the application of DNA size selection analysis for fetaltrisomy 21 detection with a plot of the z-scores for chromosome 21 ateach DNA size cutoff according to an embodiment of the presentinvention.

FIG. 18B is a histogram showing the coefficient of variation ofmeasuring the proportion of retained chr21 reads at each size cutoffusing the euploid cases according to an embodiment of the presentinvention.

DEFINITIONS

The term “biological sample” as used herein refers to any sample that istaken from a subject (e.g., a human, such as a pregnant woman) andcontains one or more nucleic acid molecule(s) of interest.

The term “nucleic acid” or “polynucleotide” refers to a deoxyribonucleicacid (DNA) or ribonucleic acid (RNA) and a polymer thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, small noncoding RNA, micro RNA(miRNA), Piwi-interacting RNA, and short hairpin RNA (shRNA) encoded bya gene or locus.

The term “gene” means the segment of DNA involved in producing apolypeptide chain. It may include regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “reaction” as used herein refers to any process involving achemical, enzymatic, or physical action that is indicative of thepresence or absence of a particular polynucleotide sequence of interest.An example of a “reaction” is an amplification reaction such as apolymerase chain reaction (PCR). Another example of a “reaction” is asequencing reaction, either by synthesis or by ligation. An “informativereaction” is one that indicates the presence of one or more particularpolynucleotide sequence of interest, and in one case where only onesequence of interest is present. The term “well” as used herein refersto a reaction at a predetermined location within a confined structure,e.g., a well-shaped vial, cell, or chamber in a PCR array.

The term “clinically relevant nucleic acid sequence” as used herein canrefer to a polynucleotide sequence corresponding to a segment of alarger genomic sequence whose potential imbalance is being tested or tothe larger genomic sequence itself. One example is the sequence ofchromosome 21. Other examples include chromosome 18, 13, X and Y. Yetother examples include mutated genetic sequences or geneticpolymorphisms or copy number variations that a fetus may inherit fromone or both of its parents. Yet other examples include sequences whichare mutated, deleted, or amplified in a malignant tumor, e.g. sequencesin which loss of heterozygosity or gene duplication occur. In someembodiments, multiple clinically relevant nucleic acid sequences, orequivalently multiple makers of the clinically relevant nucleic acidsequence, can be used to provide data for detecting the imbalance. Forinstance, data from five non-consecutive sequences on chromosome 21 canbe used in an additive fashion for the determination of possiblechromosomal 21 imbalance, effectively reducing the need of sample volumeto 1/5.

The term “background nucleic acid sequence” as used herein may refer tonucleic acid sequences originating from the mother or originating from achromosome not tested for aneuploidy in a particular analysis, which canbe, e.g., a bioinformatic one, or one involving laboratory work, or acombination.

The term “reference nucleic acid sequence” as used herein refers to anucleic acid sequence whose average concentration per reaction is knownor equivalently has been measured.

The term “overrepresented nucleic acid sequence” as used herein refersto the nucleic acid sequence among two sequences of interest (e.g., aclinically relevant sequence and a background sequence) that is in moreabundance than the other sequence in a biological sample.

The term “based on” as used herein means “based at least in part on” andrefers to one value (or result) being used in the determination ofanother value, such as occurs in the relationship of an input of amethod and the output of that method. The term “derive” as used hereinalso refers to the relationship of an input of a method and the outputof that method, such as occurs when the derivation is the calculation ofa formula.

The term “quantitative data” as used herein means data that are obtainedfrom one or more reactions and that provide one or more numericalvalues. For example, the number of wells that show a fluorescent markerfor a particular sequence would be quantitative data.

The term “parameter” as used herein means a numerical value thatcharacterizes a quantitative data set and/or a numerical relationshipbetween quantitative data sets. For example, a ratio (or function of aratio) between a first amount of a first nucleic acid sequence and asecond amount of a second nucleic acid sequence is a parameter.

The term “cutoff value” as used herein means a numerical value whosevalue is used to arbitrate between two or more states (e.g. diseased andnon-diseased) of classification for a biological sample. For example, ifa parameter is greater than the cutoff value, a first classification ofthe quantitative data is made (e.g. diseased state); or if the parameteris less than the cutoff value, a different classification of thequantitative data is made (e.g. non-diseased state).

The term “imbalance” as used herein means any significant deviation asdefined by at least one cutoff value in a quantity of the clinicallyrelevant nucleic acid sequence from a reference quantity. For example,the reference quantity could be a ratio of 3/5, and thus an imbalancewould occur if the measured ratio is 1:1.

The term “chromosomal aneuploidy” as used herein means a variation inthe quantitative amount of a chromosome from that of a diploid genome.The variation may be a gain or a loss. It may involve the whole of onechromosome or a region of a chromosome.

The term “random sequencing” as used herein refers to sequencing wherebythe nucleic acid fragments sequenced have not been specificallyidentified or targeted before the sequencing procedure.Sequence-specific primers to target specific gene loci are not required.The pools of nucleic acids sequenced vary from sample to sample and evenfrom analysis to analysis for the same sample. The identities of thesequenced nucleic acids are only revealed from the sequencing outputgenerated. In some embodiments of the present invention, the randomsequencing may be preceded by procedures to enrich a biological samplewith particular populations of nucleic acid molecules sharing certaincommon features. In one embodiment, each of the fragments in thebiological sample have an equal probability of being sequenced.

The term “fraction of the human genome” or “portion of the human genome”as used herein refers to less than 100% of the nucleotide sequences inthe human genome which comprises of some 3 billion basepairs ofnucleotides. In the context of sequencing, it refers to less than 1-foldcoverage of the total nucleotide sequences in the human genome. The termmay be expressed as a percentage or absolute number ofnucleotides/basepairs. As an example of use, the term may be used torefer to the actual amount of sequencing performed. Embodiments maydetermine the required minimal value for the sequenced fraction of thehuman genome to obtain an accurate diagnosis. As another example of use,the term may refer to the amount of sequenced data used for deriving aparameter or amount for disease classification.

The term “sequenced tag” as used herein refers to string of nucleotidessequenced from any part or all of a nucleic acid molecule. For example,a sequenced tag may be a short string of nucleotides sequenced from anucleic acid fragment, a short string of nucleotides at both ends of anucleic acid fragment, or the sequencing of the entire nucleic acidfragment that exists in the biological sample. A nucleic acid fragmentis any part of a larger nucleic acid molecule. A fragment (e.g. a gene)may exist separately (i.e. not connected) to the other parts of thelarger nucleic acid molecule.

DETAILED DESCRIPTION

Embodiments of this invention provide methods, systems, and apparatusfor determining whether an increase or decrease (diseased state) of aclinically-relevant chromosomal region exists compared to a non-diseasedstate. This determination may be done by using a parameter of an amountof a clinically-relevant chromosomal region in relation to othernon-clinically-relevant chromosomal regions (background regions) withina biological sample. Nucleic acid molecules of the biological sample aresequenced, such that a fraction of the genome is sequenced, and theamount may be determined from results of the sequencing. One or morecutoff values are chosen for determining whether a change compared to areference quantity exists (i.e. an imbalance), for example, with regardsto the ratio of amounts of two chromosomal regions (or sets of regions).

The change detected in the reference quantity may be any deviation(upwards or downwards) in the relation of the clinically-relevantnucleic acid sequence to the other non-clinically-relevant sequences.Thus, the reference state may be any ratio or other quantity (e.g. otherthan a 1-1 correspondence), and a measured state signifying a change maybe any ratio or other quantity that differs from the reference quantityas determined by the one or more cutoff values.

The clinically relevant chromosomal region (also called a clinicallyrelevant nucleic acid sequence) and the background nucleic acid sequencemay come from a first type of cells and from one or more second types ofcells. For example, fetal nucleic acid sequences originating fromfetal/placental cells are present in a biological sample, such asmaternal plasma, which contains a background of maternal nucleic acidsequences originating from maternal cells. In one embodiment, the cutoffvalue is determined based at least in part on a percentage of the firsttype of cells in a biological sample. Note the percentage of fetalsequences in a sample may be determined by any fetal-derived loci andnot limited to measuring the clinically-relevant nucleic acid sequences.In another embodiment, the cutoff value is determined at least in parton the percentage of tumor sequences in a biological sample, such asplasma, serum, saliva or urine, which contains a background of nucleicacid sequences derived from the non-malignant cells within the body.

I. General Method

FIG. 1A is a flowchart of a method 100 for performing prenatal diagnosisof a fetal chromosomal aneuploidy in a biological sample obtained from apregnant female subject according to an embodiment of the presentinvention.

In step 110, a biological sample from the pregnant female is received.The biological sample may be plasma, urine, serum, or any other suitablesample. The sample contains nucleic acid molecules from the fetus andthe pregnant female. For example, the nucleic acid molecules may befragments from chromosomes.

In step 120, at least a portion of a plurality of the nucleic acidmolecules contained in the biological sample are sequenced. The portionsequenced represents a fraction of the human genome. In one embodiment,the nucleic acid molecules are fragments of respective chromosomes. Oneend (e.g. 35 basepairs (bp)), both ends, or the entire fragment may besequenced. All of the nucleic acid molecules in the sample may besequenced, or just a subset may be sequenced. This subset may berandomly chosen, as will be described in more detail later.

In one embodiment, the sequencing is done using massively parallelsequencing. Massively parallel sequencing, such as that achievable onthe 454 platform (Roche) (Margulies, M. et al. 2005 Nature 437,376-380), Illumina Genome Analyzer (or Solexa platform) or SOLiD System(Applied Biosystems) or the Helicos True Single Molecule DNA sequencingtechnology (Harris TD et al. 2008 Science, 320, 106-109), the singlemolecule, real-time (SMRT™) technology of Pacific Biosciences, andnanopore sequencing (Soni GV and Meller A. 2007 Clin Chem 53:1996-2001), allow the sequencing of many nucleic acid molecules isolatedfrom a specimen at high orders of multiplexing in a parallel fashion(Dear Brief Funct Genomic Proteomic 2003; 1: 397-416). Each of theseplatforms sequences clonally expanded or even non-amplified singlemolecules of nucleic acid fragments.

As a high number of sequencing reads, in the order of hundred thousandsto millions or even possibly hundreds of millions or billions, aregenerated from each sample in each run, the resultant sequenced readsform a representative profile of the mix of nucleic acid species in theoriginal specimen. For example, the haplotype, trascriptome andmethylation profiles of the sequenced reads resemble those of theoriginal specimen (Brenner et al Nat Biotech 2000; 18: 630-634; Tayloret al Cancer Res 2007; 67: 8511-8518). Due to the large sampling ofsequences from each specimen, the number of identical sequences, such asthat generated from the sequencing of a nucleic acid pool at severalfolds of coverage or high redundancy, is also a good quantitativerepresentation of the count of a particular nucleic acid species orlocus in the original sample.

In step 130, based on the sequencing (e.g. data from the sequencing), afirst amount of a first chromosome (e.g. the clinically relevantchromosome) is determined. The first amount is determined from sequencesidentified as originating from the first chromosome. For example, abioinformatics procedure may then be used to locate each of these DNAsequences to the human genome. It is possible that a proportion of suchsequences will be discarded from subsequent analysis because they arepresent in the repeat regions of the human genome, or in regionssubjected to inter-individual variations, e.g. copy number variations.An amount of the chromosome of interest and of one or more otherchromosomes may thus be determined.

In step 140, based on the sequencing, a second amount of one or moresecond chromosomes is determined from sequences identified asoriginating from one of the second chromosomes. In one embodiment, thesecond chromosomes are all of the other chromosomes besides the firstone (i.e. the one being tested). In another embodiment, the secondchromosome is just a single other chromosome.

There are a number of ways of determining the amounts of thechromosomes, including but not limited to counting the number ofsequenced tags, the number of sequenced nucleotides (basepairs) or theaccumulated lengths of sequenced nucleotides (basepairs) originatingfrom particular chromosome(s) or chromosomal regions.

In another embodiment, rules may be imposed on the results of thesequencing to determine what gets counted. In one aspect, an amount maybe obtained based on a proportion of the sequenced output. For example,sequencing output corresponding to nucleic acid fragments of a specifiedsize range could be selected after the bioinformatics analysis. Examplesof the size ranges are about <300 bp, <200 bp or <100 bp. Other examplesinclude ranges of less than other values, such as 255 bp or other valuesbetween 300 bp to 50 bp.

In step 150, a parameter is determined from the first amount and thesecond amount. The parameter may be, for example, a simple ratio of thefirst amount to the second amount, or the first amount to the secondamount plus the first amount. In one aspect, each amount could be anargument to a function or separate functions, where a ratio may be thentaken of these separate functions. One skilled in the art willappreciate the number of different suitable parameters.

In one embodiment, a parameter (e.g. a fractional representation of theclinically-relevant nucleic acids to the background nucleic acids) of achromosome potentially involved in a chromosomal aneuploidy, e.g.chromosome 21 or chromosome 18 or chromosome 13, may then be calculatedfrom the results of the bioinformatics procedure. The fractionalrepresentation may be obtained based on an amount of all of thesequences (e.g. some measure of all of the chromosomes including theclinically-relevant chromosome) or a particular subset of chromosomes(e.g. just one other chromosome than the one being tested.)

In step 150, the parameter is compared to one or more cutoff values. Thecutoff values may be determined from any number of suitable ways. Suchways include Bayesian-type likelihood method, sequential probabilityratio testing (SPRT), false discovery, confidence interval, receiveroperating characteristic (ROC). Examples of applications of thesemethods and sample-specific methods are described in concurrently filedapplication “DETERMINING A NUCLEIC ACID SEQUENCE IMBALANCE,” (AttorneyDocket No. 016285-005210US), which is incorporated by reference.

In one embodiment, the parameter (e.g. the fractional representation ofthe clinically relevant chromosome) is then compared to a referencerange established in pregnancies involving normal (i.e. euploid)fetuses. It is possible that in some variants of the procedure, thereference range (i.e. the cutoff values) would be adjusted in accordancewith the fractional concentration of fetal DNA (f) in a particularmaternal plasma sample. The value of f can be determined from thesequencing dataset, e.g. using sequences mappable to the Y chromosome ifthe fetus is male. The value off may also be determined in a separateanalysis, e.g. using fetal epigenetic markers (Chan KCA et al 2006 ClinChem 52, 2211-8) or from the analysis of single nucleotidepolymorphisms.

In step 160, based on the comparison, a classification of whether afetal chromosomal aneuploidy exists for the first chromosome isdetermined. In one embodiment, the classification is a definitive yes orno. In another embodiment, a classification may be unclassifiable oruncertain. In yet another embodiment, the classification may be a scorethat is to be interpreted at a later date, for example, by a doctor.

II. Sequencing, Aligning, and Determining Amounts

As mentioned above, only a fraction of the genome is sequenced. In oneaspect, a pool of nucleic acids in a specimen is sequenced at <100%genomic coverage instead of at several folds of coverage, and among theproportion of captured nucleic acid molecules, most of each nucleic acidspecies is only sequenced once. Also, dosage imbalance of a particularchromosome or chromosomal regions can be quantitatively determined. Inother words, the dosage imbalance of the chromosome or chromosomalregions is inferred from the percentage representation of the said locusamong other mappable sequenced tags of the specimen.

This is contrasted from situations where the same pool of nucleic acidsis sequenced multiple times to achieve high redundancy or several foldsof coverage whereby each nucleic acid species is sequenced multipletimes. In such situations, the number of times a particular nucleic acidspecies have been sequenced relative to that of another nucleic acidspecies correlate with their relative concentrations in the originalsample. The sequencing cost increases with the number of fold coveragerequired to achieve accurate representation of the nucleic acid species.

In one example, a proportion of such sequences would be from thechromosome involved in an aneuploidy such as chromosome 21 in thisillustrative example. Yet other sequences from such a sequencingexercise would be derived from the other chromosomes. By taking intoaccount of the relative size of chromosome 21 compared with the otherchromosomes, one could obtain a normalized frequency, within a referencerange, of chromosome 21-specific sequences from such a sequencingexercise. If the fetus has trisomy 21, then the normalized frequency ofchromosome 21-derived sequences from such a sequencing exercise willincrease, thus allowing the detection of trisomy 21. The degree ofchange in the normalized frequency will be dependent on the fractionalconcentration of fetal nucleic acids in the analyzed sample.

In one embodiment, we used the Illumina Genome Analyzer for single-endsequencing of human genomic DNA and human plasma DNA samples. TheIllumina Genome Analyzer sequences clonally-expanded single DNAmolecules captured on a solid surface termed a flow cell. Each flow cellhas 8 lanes for the sequencing of 8 individual specimens or pools ofspecimens. Each lane is capable of generating ˜200 Mb of sequence whichis only a fraction of the 3 billion basepairs of sequences in the humangenome. Each genomic DNA or plasma DNA sample was sequenced using onelane of a flow cell. The short sequence tags generated were aligned tothe human reference genome sequence and the chromosomal origin wasnoted. In one embodiment, perfect alignment is not required. The totalnumber of individual sequenced tags aligned to each chromosome weretabulated and compared with the relative size of each chromosome asexpected from the reference human genome or non-disease representativespecimens. Chromosome gains or losses were then identified.

The described approach is only one exemplification of the presentlydescribed gene/chromosome dosage strategy. Alternatively, paired endsequencing could be performed. Instead of comparing the length of thesequenced fragments from that expected in the reference genome asdescribed by Campbell et al (Nat Genet 2008; 40: 722-729), the number ofaligned sequenced tags were counted and sorted according to chromosomallocation. Gains or losses of chromosomal regions or whole chromosomeswere determined by comparing the tag counts with the expected chromosomesize in the reference genome or that of a non-disease representativespecimen. As paired end sequencing allows one to deduce the size of theoriginal nucleic acid fragment, one example is to focus on the countingof the number of paired sequenced tags corresponding to nucleic acidfragments of a specified size, such as <300 bp, <200 bp or <100 bp.

In another embodiment, the fraction of the nucleic acid pool that issequenced in a run is further sub-selected prior to sequencing. Forexample, hybridization based techniques such as oligonucleotide arraycould be used to first sub-select for nucleic acid sequences fromcertain chromosomes, e.g. a potentially aneuploid chromosome and otherchromosome(s) not involved in the aneuploidy tested. Another example isthat a certain sub-population of nucleic acid sequences from the samplepool is sub-selected or enriched prior to sequencing. For example, ithas been reported that fetal DNA molecules in maternal plasma arecomprised of shorter fragments than the maternal background DNAmolecules (Chan et al Clin Chem 2004; 50: 88-92). Thus, one may use oneor more methods known to those of skill in the art to fractionate thenucleic acid sequences in the sample according to molecule size, e.g. bygel electrophoresis or size exclusion columns or by microfluidics-basedapproach. Yet, alternatively, in the example of analyzing cell-freefetal DNA in maternal plasma, the fetal nucleic acid portion could beenriched by a method that suppresses the maternal background, such as bythe addition of formaldehyde (Dhallan et al JAMA 2004; 291: 1114-9). Inone embodiment, a portion or subset of the pre-selected pool of nucleicacids is sequenced randomly.

Other single molecule sequencing strategies such as that by the Roche454 platform, the Applied Biosystems SOLiD platform, the Helicos TrueSingle Molecule DNA sequencing technology, the single molecule,real-time (SMRT™) technology of Pacific Biosciences, and nanoporesequencing could similarly be used in this application.

III. Determining Amounts of Chromosomes from Sequencing Output

After the massively parallel sequencing, bioinformatics analysis wasperformed to locate the chromosomal origin of the sequenced tags. Afterthis procedure, tags identified as originating from the potentiallyaneuploid chromosome, i.e. chromosome 21 in this study, are comparedquantitatively to all of the sequenced tags or tags originating from oneof more chromosomes not involved in the aneuploidy. The relationshipbetween the sequencing output from chromosome 21 and other non-21chromosomes for a test specimen is compared with cut-off values derivedwith methods described in the above section to determine if the specimenwas obtained from a pregnancy involving a euploid or trisomy 21 fetus.

A number of different amounts include but not limited to the followingcould be derived from the sequenced tags. For example, the number ofsequenced tags, i.e. absolute count, aligned to a particular chromosomecould be compared to the absolute count of sequenced tags aligned toother chromosomes. Alternatively, the fractional count of the amount ofsequenced tags from chromosome 21 with reference to all or some othersequenced tags could be compared to that of other non-aneuploidchromosomes. In the present experiment, because 36 bp were sequencedfrom each DNA fragment, the number of nucleotides sequenced from aparticular chromosome could easily be derived from 36 bp multiplied bythe sequenced tag count.

Furthermore, as each maternal plasma specimen was only sequenced usingone flow cell which could only sequence a fraction of the human genome,by statistics, most of the maternal plasma DNA fragment species wouldonly each have been sequenced to generate one sequenced tag count. Inother words, the nucleic acid fragments present in the maternal plasmaspecimen were sequenced at less than 1-fold coverage. Thus, the totalnumber of sequenced nucleotides for any particular chromosome wouldmostly correspond to the amount, proportion or length of the part of thesaid chromosome that has been sequenced. Hence, the quantitativedetermination of the representation of the potentially aneuploidchromosome could be derived from a fraction of the number or equivalentlength of nucleotides sequenced from that chromosome with reference to asimilarly derived quantity for other chromosomes.

Counting Based on Length

As described in examples II and IV below, a subset of the sequenced datais sufficient to distinguish trisomy 21 from euploid cases. The subsetof sequenced data could be the proportion of sequenced tags that passedcertain quality parameters. For example, in example II, sequenced tagsthat were uniquely aligned to the repeat-masked reference human genomewere used. Alternatively, one may sequence a representative pool ofnucleic acid fragments from all of the chromosomes but focus on thecomparison between data relevant to the potentially aneuploid chromosomeand data relevant to a number of non-aneuploid chromosomes. Yetalternatively, a subset of the sequencing output encompassing sequencedtags generated from nucleic acid fragments corresponding to a specifiedsize window in the original specimen could be sub-selected during thepost-sequencing analysis.

FIG. 1B is a flowchart of another method 101 for performing prenataldiagnosis of a fetal chromosomal aneuploidy in a biological sampleobtained from a pregnant female subject according to an embodiment ofthe present invention.

In step 111, a biological sample from the pregnant female is received.The sample contains nucleic acid molecules from the fetus and thepregnant female. For example, the nucleic acid molecules may befragments from chromosomes.

In step 121, at least a portion of a plurality of the nucleic acidmolecules contained in the biological sample are sequenced. In oneembodiment, the nucleic acid molecules are fragments of respectivechromosomes. At least both ends of the fragments are sequenced, and theentire fragment may be sequenced. All of the nucleic acid molecules inthe sample may be sequenced, or just a subset may be sequenced. Thissubset may be randomly chosen, as will be described in more detaillater. In one embodiment, the Illumina Genome analyzer is used toperform the paired-end sequencing to sequence the two ends of nucleicacid fragments.

In step 131, the length of each fragment is determined. In oneembodiment, the sequenced data from each paired-end are aligned to areference sequence (e.g. the reference human genome sequence), e.g.,using BLAST. The distance or number of nucleotides spanning between thetwo ends is then determined to be the length of the sequenced fragment.Effectively, the whole fragment is sequenced by performing thealignment. Thus, the length of the sequences of the fragment is deduced.

Alternatively, sequencing platforms such as the 454 platform andpossibly some single molecule sequencing techniques are able to sequencethe full length of short nucleic acid fragments, for example 200 bp orequivalently nucleotides (nt). In this manner, the actual length of thenucleic acid fragment would be immediately known from the sequenceddata.

Such paired-end analysis is also possible using other sequencingplatforms, e.g. the Applied Biosystems SOLiD system. For the Roche 454platform, because of its increased read length compared with othermassively parallel sequencing systems, it is also possible to determinethe length of a fragment from its complete sequence.

In step 141, based on the sequencing and the lengths, a first amount ofa first chromosome is determined. In one embodiment, fragments of aspecified length are counted. The specified length may be a specificnumber of nucleotides (or base pairs) or a range of lengths. Forexample, the length may be specified to be greater than or less than anumber, or greater (less) than or equal to the number. As anotherexample, the range may specified to be between two numbers, andoptionally including the numbers.

The counted fragments are associated with a particular chromosome, forexample, as may be done during the alignment procedure. The first amountis thus determined from sequences that are identified as originatingfrom the first chromosome and that are of the specified length. In oneaspect, a proportion of such counted sequences may be discarded from theamount because they are present in the repeat regions of the humangenome, or in regions subjected to inter-individual variations, e.g.copy number variations.

In step 151, based on the sequencing and the lengths, a second amount ofone or more second chromosomes is determined from sequences identifiedas originating from one of the second chromosomes. In one embodiment,the specified length for the fragments counted to determine the secondamount is a different specified length than the specified length used todetermine the first amount, as in step 141. Different lengths may beused for different chromosomes as well. All of the ways of determiningthe amounts from method 100 may also be used for method 100.

In step 151, a parameter is determined from the first amount and thesecond amount. The parameter may be as described for method 100 andother places herein. In step 161, the parameter is compared to one ormore cutoff values. The cutoff values may be determined from any numberof suitable ways, as in method 100 and other places herein.

In step 171, based on the comparison, a classification of whether afetal chromosomal aneuploidy exists for the first chromosome isdetermined. In one embodiment, the classification is a definitive yes orno. In another embodiment, a classification may be unclassifiable oruncertain. In yet another embodiment, the classification may be a scorethat is to be interpreted at a later date, for example, by a doctor.

In further embodiments, certain chromosomes may be selected to be usedas the second chromosomes for determining the second amount and theparameter that is compared to the cutoff values. For example, the secondchromosomes may be selected to have similar properties as the firstchromosome. In one embodiment, the second chromosomes are selected suchthat the nucleic acid molecules (i.e. fragments) of the one or moresecond chromosomes have an expected average length that is within twonucleotides of the expected average length for the first chromosome. Inanother embodiment, the nucleic acid molecules of the one or more secondchromosomes have an expected maximum and minimum length that are bothwithin two nucleotides of the expected maximum and minimum length forthe first chromosome.

In other embodiments, the second chromosomes have different propertieswhich may be accounted for. For example, the second chromosomes may havedifferent lengths of fragments than the first chromosome. The secondchromosomes may also have different properties amongst themselves. Inone embodiment, the different properties is accounted for by selectingsequences that originate from at least one of the second chromosomes tobe of a different specified length (e.g. less than a different number ofnt) than the sequences of the first chromosome. In one aspect, thedifferent specified length is selected based on an expected sizedistribution for the nucleic acid molecules of the least one of thesecond chromosomes that are in the biological sample.

In one embodiment, the specified length for counting focuses on shortnucleic acid fragments. An advantage of focusing the data analysis onthe subset of sequenced tags corresponding to short nucleic acidfragments in the original maternal plasma specimen is because thedataset would effectively be enriched with DNA sequences derived fromthe fetus. This is because the fetal DNA molecules in maternal plasmaare comprised of shorter fragments than the maternal background DNAmolecules (Chan et al Clin Chem 2004; 50: 88-92).

According to FIG. 7, the number of sequenced tags required fordifferentiating euploid from trisomy 21 cases would reduce as thefractional fetal DNA concentration increases. However, the increase ofthe fractional fetal DNA concentration by counting only smallerfragments comes at a cost of providing a fewer number of totalsequences, which can cause statistical fluctuations and errors in theclassification, e.g., false positives. Accordingly, in one embodiment,the one or more specified lengths (potentially for different chromosomesper above) for counting the fragments are selected to provide at least aspecific total amount for the first amount and the second amount. Indifferent embodiments, the total amount is two million, 1 million,500,000, or 250,000.

Also, the one or more specified lengths for counting the fragments maybe selected to balance an increase in the percentage of sequenced fetalfragments and the total number of sequences. In an embodiment wheresequences from the first chromosome are counted if they are less than afirst predetermined number of nucleotides, the first specified number ofnucleotides selected to be between about 125 nucleotides and about 175nucleotides. Such a selection can provide such a balance.

In another embodiment, the sequences that originate from the firstchromosome are selected to be greater than a second specified number ofnucleotides. In one aspect, the second specified number is between 100and 125 nucleotides. In another aspect, such a minimum length canprovide for greater accuracy. For example, a benefit is to remove theultra-short fragments that are generated in vitro, e.g., by DNAdegradation, instead of genuinely present in plasma in the in vivostate. The sequences that originate from at least one of the secondchromosomes may also selected to be greater than a specified number ofnucleotides, which may be different than the second specified number.Example II below provides data regarding optimal lengths to be used forcounting the fragments.

As another advantage, the post-sequencing selection of subsets ofnucleic acid pools is different from other nucleic acid enrichmentstrategies which are performed prior to specimen analysis, such as theuse gel electrophoresis or size exclusion columns for the selection ofnucleic acids of particular sizes, which require the physical separationof the enriched pool from the background pool of nucleic acids. Thephysical procedures would introduce more experimental steps and may beprone to problems such as contamination. The post-sequencing in silicoselection of subsets of sequencing output would also allow one to varythe selection depending on the sensitivity and specificity required fordisease determination.

IV. Enrichment for Pools of Nucleic Acids for Sequencing

As mentioned above and established in the example section below, only aportion of the human genome needs to be sequenced to differentiatetrisomy 21 from euploid cases. Thus, it would be possible andcost-effective to enrich the pool of nucleic acids to be sequenced priorto random sequencing of a fraction of the enriched pool. For example,fetal DNA molecules in maternal plasma are comprised of shorterfragments than the maternal background DNA molecules (Chan et al ClinChem 2004; 50: 88-92). Thus, one may use one or more methods known tothose of skill in the art to fractionate the nucleic acid sequences inthe sample according to molecule size (e.g. by number of nucleotides),e.g. by gel electrophoresis (Li et al Clin Chem 2004; 50: 1002-1011) orsize exclusion columns or by microfluidics-based approach. The specificsizes (e.g. lengths) chosen for the fractionating may be the samespecified lengths used for the counting described above.

Yet, alternatively, in the example of analyzing cell-free fetal DNA inmaternal plasma, the fetal nucleic acid portion could be enriched by amethod that suppresses the maternal background, such as by the additionof formaldehyde (Dhallan et al JAMA 2004; 291: 1114-9). The proportionof fetal derived sequences would be enriched in the nucleic acid poolcomprised of shorter fragments. According to FIG. 7, the number ofsequenced tags required for differentiating euploid from trisomy 21cases would reduce as the fractional fetal DNA concentration increases.

Alternatively, sequences originating from a potentially aneuploidchromosome and one or more chromosomes not involved in the aneuploidycould be enriched by hybridization techniques for example ontooligonucleotide microarrays. Examples of commercially available productsfor allowing such enrichment by hybridization includes the NimbleGenSequence Capture microarrays and Agilent SureSelect Target EnrichmentSystem. The enriched pools of nucleic acids would then be subjected torandom sequencing. This would allow the reduction in sequencing costs.

V. Random Sequencing

FIG. 2 is a flowchart of a method 200 for performing prenatal diagnosisof a fetal chromosomal aneuploidy using random sequencing according toan embodiment of the present invention. In one aspect for the massivelyparallel sequencing approach, representative data from all of thechromosomes may be generated at the same time. The origin of aparticular fragment is not selected ahead of time. The sequencing isdone at random and then a database search may be performed to see wherea particular fragment is coming from. This is contrasted from situationswhen a specific fragment from chromosome 21 and another one fromchromosome 1 are amplified.

In step 210, a biological sample from the pregnant female is received.In step 220, the number N of sequences to be analyzed is calculated fora desired accuracy. In one embodiment, a percentage of fetal DNA in thebiological sample is first identified. This may be done by any suitablemeans as will be known to one skilled in the art. The identification maysimply be reading a value that was measured by another entity. In thisembodiment, the calculation of the number N of sequences to be analyzedis based on the percentage. For example, the number of sequences neededto be analyzed would be increased when the fetal DNA percentage drops,and could be decreased when the fetal DNA rises. The number N may be afixed number or a relative number, such as a percentage. In anotherembodiment, one could sequence a number N that is known to be adequatefor accurate disease diagnosis. The number N could be made sufficienteven in pregnancies with fetal DNA concentrations that are at the lowerend of the normal range.

In step 230, at least N of a plurality of the nucleic acid moleculescontained in the biological sample are randomly sequenced. A feature ofthis described approach is that the nucleic acids to be sequenced arenot specifically identified or targeted before sample analysis, i.e.sequencing. Sequence-specific primers to target specific gene loci arenot needed for sequencing. The pools of nucleic acids sequenced varyfrom sample to sample and even from analysis to analysis for the samesample. Furthermore, from the below descriptions, the amount ofsequencing output required for case diagnosis could vary between thetested specimens and the reference population. These aspects are inmarked contrast to most molecular diagnostic approaches, such as thosebased on fluorescence in situ hybridization, quantitative florescencePCR, quantitative real-time PCR, digital PCR, comparative genomichybridization, microarray comparative genomic hybridization and so on,where gene loci to be targeted require prior pre-determination, thusrequiring the use of locus-specific primers or probe sets or panels ofsuch.

In one embodiment, random sequencing is performed on DNA fragments thatare present in the plasma of a pregnant woman, and one obtains genomicsequences which would originally have come from either the fetus or themother. Random sequencing involves sampling (sequencing) a randomportion of the nucleic acid molecules present in the biological sample.As the sequencing is random, a different subset (fraction) of thenucleic acid molecules (and thus the genome) may be sequenced in eachanalysis. Embodiments will work even when this subset varies from sampleto sample and from analysis to analysis, which may occur even using thesame sample. Examples of the fraction are about 0.1%, 0.5%, 1%, 5%, 10%,20%, or 30% of the genome. In other embodiments, the fraction is atleast any one of these values.

The rest of the steps 240-270 may proceed in a similar manner as method100.

Regarding the use (or non-use) of a locus to identify an identity of afragment, FIG. 10 shows a schematic comparison between locus-specificand locus-independent methods (e.g. methods described herein) for DNAquantification.

DNA molecules exist as short fragments in maternal plasma. Hence,instead of comparing the relative amounts between specific loci as withconventional DNA quantification methods, the amount of quantitativeinformation that one could derive with the same amount of plasma DNAinput greatly increases with the use of locus-independent quantificationmethods that treat each DNA fragment as an individual target. Forexample, as depicted in FIG. 10 a, when using locus-specific assays,five copies of chromosome 21 with the targeted amplicon region intactwould be needed to be physically present to generate a count of five.However, as shown in FIG. 10 b, in the locus-independent method, fivefragmented portions originating from a single chromosome 21 couldpotentially contribute to a count of five.

The bioinformatics, computational and statistical approaches used todetermine if a maternal plasma specimen is obtained from a pregnantwoman conceived with a trisomy 21 or euploid fetus could be compiledinto a computer program product used to determine parameters from thesequencing output. The operation of the computer program would involvethe determining of a quantitative amount from the potentially aneuploidchromosome as well as amount(s) from one or more of the otherchromosomes. A parameter would be determined and compared withappropriate cut-off values to determine if a fetal chromosomalaneuploidy exists for the potentially aneuploid chromosome.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

I. Prenatal Diagnosis of Fetal Trisomy 21

Eight pregnant women were recruited for the study. All of the pregnantwomen were in the 1^(st) or 2^(nd) trimester of gestation and had asingleton pregnancy. Four of them were each carrying a fetus withtrisomy 21 and the other four were each carrying a euploid fetus. Twentymilliliters of peripheral venous blood were collected from each subject.Maternal plasma was harvested after centrifugation at 1600×g for 10minutes and further centrifuged at 16000×g for 10 minutes. DNA was thenextracted from 5-10 mL of each plasma sample. The maternal plasma DNAwas then used for massively parallel sequencing by the Illumina GenomeAnalyzer according to manufacturer's instructions. The techniciansperforming the sequencing were blinded from the fetal diagnoses duringthe sequencing and sequence data analysis.

Briefly, approximately 50 ng of maternal plasma DNA was used for DNAlibrary preparation. It is possible to start with lesser amounts such as15 ng or 10 ng of maternal plasma DNA. Maternal plasma DNA fragmentswere blunt-ended, ligated to Solexa adaptors and fragments of 150-300 bpwere selected by gel purification. Alternatively, blunt-ended andadaptor-ligated maternal plasma DNA fragments could be passed throughcolumns (e.g. AMPure, Agencourt) to remove unligated adaptors withoutsize-selection before cluster generation. The adaptor-ligated DNA washybridized to the surface of flow cells, and DNA clusters were generatedusing the Illumina cluster station, followed by 36 cycles of sequencingon the Illumina Genome Analyzer. DNA from each maternal plasma specimenwas sequenced by one flow cell. Sequenced reads were compiled usingSolexa Analysis Pipeline. All reads were then aligned to therepeat-masked reference human genomic sequence, NCBI 36 assembly(GenBank accession numbers: NC_(—)000001 to NC_(—)000024), using theEland application.

In this study, to reduce the complexity of the data analysis, onlysequences that have been mapped to a unique location in therepeat-masked human genome reference are further considered. Othersubsets of or the entire set of the sequenced data could alternativelybe used. The total number of uniquely mappable sequences for eachspecimen was counted. The number of sequences uniquely aligned tochromosome 21 was expressed as a proportion to the total count ofaligned sequences for each specimen. As maternal plasma contains fetalDNA among a background of DNA of maternal origin, the trisomy 21 fetuswould contribute extra sequenced tags originating from chromosome 21 dueto the presence of an extra copy of chromosome 21 in the fetal genome.Hence, the percentage of chromosome 21 sequences in maternal plasma froma pregnancy carrying a trisomy 21 fetus would be higher than that from apregnancy with a euploid fetus. The analysis does not require thetargeting of fetal-specific sequences. It also does not require theprior physical separation of fetal from maternal nucleic acids. It alsodoes not require the need to distinguish or identify fetal from maternalsequences after sequencing.

FIG. 3A shows the percentage of sequences mapped to chromosome 21(percentage representation of chromosome 21) for each of the 8 maternalplasma DNA samples. The percentage representation of chromosome 21 wassignificantly higher in maternal plasma of trisomy 21 pregnancies thanin that of euploid pregnancies. These data suggest that noninvasiveprenatal diagnosis of fetal aneuploidy could be achieved by determiningthe percentage representation of the aneuploid chromosome compared tothat of a reference population. Alternatively, the chromosome 21over-representation could be detected by comparing the percentagerepresentation of chromosome 21 obtained experimentally with thepercentage representation of chromosome 21 sequences expected for aeuploid human genome. This could be done by masking or not masking therepeat regions in the human genome.

Five of the eight pregnant women were each carrying a male fetus. Thesequences mapped to the Y chromosome would be fetal-specific. Thepercentage of sequences mapped to the Y-chromosome was used to calculatethe fractional fetal DNA concentration in the original maternal plasmaspecimen. Moreover, the fractional fetal DNA concentration was alsodetermined by using microfluidics digital PCR involving the zinc fingerprotein, X-linked (ZFX) and zinc finger protein, Y-linked (ZFY)paralogous genes.

FIG. 3B shows the correlation of the fractional fetal DNA concentrationsas inferred by the percentage representation of Y chromosome bysequencing and that determined by ZFY/ZFXmicrofluidics digital PCR.There was a positive correlation between the fractional fetal DNAconcentrations in maternal plasma determined by these two methods. Thecoefficient of correlation (r) was 0.917 in the Pearson correlationanalysis.

The percentages of maternal plasma DNA sequences aligned to each of the24 chromosomes (22 autosomes and X and Y chromosomes) for tworepresentative cases are shown in FIG. 4A. One pregnant woman wascarrying a trisomy 21 fetus and the other was carrying a euploid fetus.The percentage representation of sequences mapped to chromosome 21 ishigher in the pregnant woman carrying a trisomy 21 fetus when comparedwith the pregnant woman carrying a normal fetus.

The differences (%) of the percentage representation per chromosomebetween the maternal plasma DNA specimens of the above two cases isshown in FIG. 4B. The percentage difference for a particular chromosomeis calculated using the formula below:

Percentage difference(%)=(P ₂₁ −P _(E))/P _(E)×100%,where

P₂₁=percentage of plasma DNA sequences aligned to the particularchromosome in the pregnant woman carrying a trisomy 21 fetus and;P_(E)=percentage of plasma DNA sequences aligned to the particularchromosome in the pregnant woman carrying a euploid fetus.

As shown in FIG. 4B, there is an over-representation of chromosome 21sequences by 11% in the plasma of the pregnant woman carrying a trisomy21 fetus when compared with the pregnant woman carrying a euploid fetus.For the sequences aligned to other chromosomes, the differences betweenthe two cases were within 5%. As the percentage representation forchromosome 21 is increased in the trisomy 21 compared with the euploidmaternal plasma samples, the difference (%) could be alternativelyreferred as the degree of over-representation in chromosome 21sequences. In addition to differences (%) and absolute differencesbetween the chromosome 21 percentage representation, ratios of thecounts from test and reference samples could also be calculated andwould be indicative of the degree of chromosome 21 over-representationin trisomy 21 compared with euploid samples.

For the four pregnant women each carrying a euploid fetus, a mean of1.345% of their plasma DNA sequences were aligned to chromosome 21. Inthe four pregnant women carrying a trisomy 21 fetus, three of theirfetuses were males. The percentage representation of chromosome 21 wascalculated for each of these three cases. The difference (%) inchromosome 21 percentage representation for each of these three trisomy21 cases from the mean chromosome 21 percentage representation derivedfrom values of the four euploid cases were determined as describedabove. In other words, the mean of the four cases carrying a euploidfetus was used as the reference in this calculation. The fractionalfetal DNA concentrations for these three male trisomy 21 cases wereinferred from their respective percentage representation of Y chromosomesequences.

The correlation between the degree of over-representation for chromosome21 sequences and the fractional fetal DNA concentrations is shown inFIG. 5. There was a significant positive correlation between the twoparameters. The coefficient of correlation (r) was 0.898 in the Pearsoncorrelation analysis. These results indicate that the degree ofover-representation of chromosome 21 sequences in maternal plasma isrelated to the fractional concentration of fetal DNA in the maternalplasma sample. Thus, cut-off values in the degree of chromosome 21sequence over-representation relevant to the fractional fetal DNAconcentrations could be determined to identify pregnancies involvingtrisomy 21 fetuses.

The determination of the fractional concentration of fetal DNA inmaternal plasma can also be done separate to the sequencing run. Forexample, the Y chromosome DNA concentration could be pre-determinedusing real-time PCR, microfluidics PCR or mass spectrometry. Forexample, we have demonstrated in FIG. 3B that there is good correlationbetween the fetal DNA concentrations estimated based on the Y-chromosomecount generated during the sequencing run and the ZFY/ZFX ratiogenerated external to the sequencing run. In fact, fetal DNAconcentration could be determined using loci other than the Y chromosomeand applicable to female fetuses. For example, Chan et al showed thatfetal-derived methylated RASSF1A sequences would be detected in theplasma of pregnant women in the background of maternally derivedunmethylated RASSF1A sequences (Chan et al, Clin Chem 2006; 52:2211-8).The fractional fetal DNA concentration can thus be determined bydividing the amount of methylated RASSF1A sequences by the amount oftotal RASSF1A (methylated and unmethylated) sequences.

It is expected that maternal plasma would be preferred over maternalserum for practicing our invention because DNA is released from thematernal blood cells during blood clotting. Thus, if serum is used, itis expected that the fractional concentration of fetal DNA will be lowerin maternal plasma than maternal serum. In other words, if maternalserum is used, it is expected that more sequences would need to begenerated for fetal chromosomal aneuploidy to be diagnosed, whencompared with a plasma sample obtained from the same pregnant woman atthe same time.

Yet another alternative way of determining the fractional concentrationof fetal DNA would be through the quantification of polymorphicdifferences between the pregnant women and the fetus (Dhallan R, et al.2007 Lancet, 369, 474-481). An example of this method would be to targetpolymorphic sites at which the pregnant woman is homozygous and thefetus is heterozygous. The amount of fetal-specific allele can becompared with the amount of the common allele to determine thefractional concentration of fetal DNA.

In contrast to the existing techniques for detecting chromosomalaberrations, including comparative genomic hybridization, microarraycomparative genomic hybridization, quantitative real-time polymerasechain reaction, which detect and quantify one or more specificsequence(s), massively parallel sequencing is not dependent on thedetection or analysis of predetermined or a predefined set of DNAsequences. A random representative fraction of DNA molecules from thespecimen pool is sequenced. The number of different sequenced tagsaligned to various chromosomal regions is compared between specimenscontaining or not containing the DNA species of interest. Chromosomalaberrations would be revealed by differences in the number (orpercentage) of sequences aligned to any given chromosomal region in thespecimens.

In another example the sequencing technique on plasma cell-free DNA maybe used to detect the chromosomal aberrations in the plasma DNA for thedetection of a specific cancer. Different cancers have a set of typicalchromosomal aberrations. Changes (amplifications and deletions) inmultiple chromosomal regions may be used. Thus, there would be anincreased proportion of sequences aligned to the amplified regions and adecreased proportion of sequences aligned to decreased regions. Thepercentage representation per chromosome could be compared with the sizefor each corresponding chromosome in a reference genome expressed aspercentage of genomic representation of any given chromosome in relationto the whole genome. Direct comparisons or comparisons to a referencechromosome may also be used.

In another example, DNA from 5 mL to 10 mL plasma from 14 trisomy 21 and14 euploid pregnancies was used. A random representative portion of DNAmolecules in the maternal plasma is sequenced. A mean of ˜2 millionunique reads per sample was obtained, without mismatches (mismatches of1 or 2 may also be allowed) to the reference human genome. Thechromosomal origin of each sequenced read is identified bybioinformatics analysis.

The mean and standard deviation of the proportion of reads from eachchromosome of a reference sample set comprising pregnancies with euploidmale fetuses are determined. Z-scores, representing the number ofstandard deviations from the mean of the reference sample set, of thepercentage chromosomal representation for each maternal plasma samplewas calculated. The number of reads originating from chromosome 21 wasexpressed as a proportion of all sequenced reads, and z-scores,representing the number of standard deviations away from the meanproportion of chromosome 21 reads in a reference set of euploid cases,were determined for each case.

FIG. 11 shows plots of z-scores for each chromosome for maternal plasmasamples from 14 trisomy 21 and 14 euploid pregnancies are shown. Thedifferent types (i.e. euploid or trisomy) of samples are categorized.Each of the 28 bars shown for each chromosome corresponded to thez-scores for one of the 28 maternal plasma samples. Samples 1 to 28 areshown consecutively from left to right.

A z-score larger than ±3 indicated a 99% chance of a statisticallysignificant difference in the assessed parameter for the test casecompared with the reference group (e.g. presence of chromosomal over- orunderrepresentation compared with the reference group). Thus, a highz-score was expected for trisomy 21 cases. The massively parallelsequencing approach was reliable and robust: in all cases, z-scoressmaller than ±3 were obtained for all chromosomes except 21 and X (FIG.11). Z-scores of chromosome 21 were beyond +5 for all 14 trisomy 21cases but within ±3 for all euploid cases. Because pregnancies with malefetuses were used as the reference sample set, z-scores for theX-chromosome were increased in all pregnancies with female fetuses.

II. Sequencing Just a Fraction of the Human Genome

In the experiment described in example I above, maternal plasma DNA fromeach individual specimen was sequenced using one flow cell only. Thenumber of sequenced tags generated from each of the tested specimens bythe sequencing run is shown in FIG. 6. T21 denotes a sample obtainedfrom a pregnancy involving a trisomy 21 fetus.

As 36 bp were sequenced from each of the sequenced maternal plasma DNAfragments, the number of nucleotides/basepairs sequenced from eachspecimen could be determined by 36 bp multiplied by the sequenced tagcount and are also shown in FIG. 6. As there are approximately 3 billionbasepairs in the human genome, the amount of sequencing data generatedfrom each maternal plasma specimen represented only a fraction, rangingfrom some 10% to 13%.

Furthermore, in this study, only the uniquely mappable sequenced tags,termed U0 in nomenclature from the Efficient Large-Scale Alignment ofNucleotide Databases (ELAND) software, were used to demonstrate thepresence of over-representation in the amount of chromosome 21 sequencesin the maternal plasma specimens from pregnancies each carrying a fetuswith trisomy 21, as described in example I above. As shown in FIG. 6, U0sequences only represent a subset of all the sequenced tags generatedfrom each specimen and further represent an even smaller proportion,some 2%, of the human genome. These data indicate that the sequencing ofonly a portion of the human genomic sequences present in the testedspecimen is sufficient to achieve the diagnosis of fetal aneuploidy.

III. Paired-End Sequencing and Length Dependent Counting

In this example, paired-end (PE) sequencing was applied directly,without fragmentation and gel electrophoresis based size selection, forsequencing of plasma from pregnancies with euploid or trisomy 21fetuses. As described above, the nucleotide length, i.e. size, of eachfragment was deduced from the alignment positions of the sequenced endsof each plasma DNA molecule.

Detection of Chromosome Dosage with Paired-End Sequencing

The ability to detect fetal DNA by paired-end sequencing is firstestablished. Placental tissue DNA from two euploid fetuses and two T21fetuses were sequenced. The proportion of accepted PE reads for eachchromosome was close to that expected for the human genome (data notshown). 1.82% and 1.85% of PE reads from chr21 were obtained from thetwo T21 placental tissue samples, respectively, which were ˜1.5-foldhigher than the proportions for the two euploid samples (1.28% and1.30%, respectively). These data suggested that the measurement ofchromosome dosage using PE sequencing was feasible.

We next sequenced three maternal plasma samples (one with a female fetusand two from pregnancies each with a male fetus) collected in the thirdtrimester.

FIG. 12 shows a bar chart of proportion of accepted PE reads for eachhuman chromosome for three maternal plasma samples collected in thethird trimester. The percentage of genomic representation of eachchromosome as expected for a repeat-masked reference haploid femalegenome was plotted for comparison (stripped bars). The percentages ofaccepted PE reads mapped to each chromosome generally resembled thegenomic representation expected for each chromosome in the human genome.Also, the absolute (and fractional) accepted PE counts mapped to chrYfor the two pregnancies with male fetuses were 710 (0.064%) and 829(0.079%), respectively, indicating positive detection of fetal DNA by PEsequencing of maternal plasma.

Sequencing and Alignment

Sequencing libraries were constructed from the extracted DNA using thePaired-End Sequencing Sample Preparation Kit (Illumina, San Diego,Calif.) mostly according to manufacturer's instructions. Since plasmaDNA molecules are short fragments by nature, we omitted the steps offragmentation and size selection by gel electrophoresis. DNA clusterswere generated using an Illumina cluster station, followed by 36×2cycles of sequencing on a Genome Analyzer II (Illumina).

The first 32 bp from the 36 bp sequenced reads were aligned to therepeat-masked human genome reference sequence (NCBI Build 36, version48) using the Efficient Large-Scale Alignment of Nucleotide Databasesfor PE sequencing (eland_pair) program (Illumina). As an illustration,we selected a subset of PE reads, namely those only PE reads meeting thefollowing criteria, termed accepted PE reads, for subsequent analysis:

1) the individual members of each suggested pair were both sequenced onthe same cluster position on the sequencing flow cell and could bealigned to the same chromosome with the correct orientation as expectedfor the reference human genome;2) the sequenced reads of both members of the pair could be aligned tothe repeat-masked reference human genome without any nucleotidemismatch;3) the sequenced reads of each member of the pair had a uniqueness score>4;4) pairs demonstrating an insert size less than 600 bp.

Approximately 1.6 million pairs (17% from a total of ˜10 millionsequenced molecules) of high quality sequenced reads, aligned uniquelyto the repeat-masked reference human genome without mismatches, wereobtained from each plasma sample. Alignment errors to chromosome Y werereduced among the accepted paired-end reads compared with unique readsobtained from single-read sequencing.

A small number of accepted PE reads were mapped to chrY in both thematernal plasma sample involving a female fetus (50 reads, 0.0047%) andthe female T21 placental tissue (64 reads, 0.0044%). Only 38% of thesesequences were confirmed by Basic Local Alignment Search Tool (BLAST)analysis to be uniquely mapped to chrY. Similarly, 150 PE reads alignedto chrY were randomly picked from each of the two plasma samples ofpregnancies with male fetuses. 90.4% (135 of 150) and 98.0% (147 of 150)of the paired sequences could be aligned uniquely to chrY by BLAST. 150paired sequences from each of the other human chromosomes were alsorandomly selected from each of the three maternal plasma samples forBLAST analysis. Almost all (98.1% for chromosomes 4 and 5, 100% for allother chromosomes) accepted PE reads mapped to the non-Y chromosomeswere validated by BLAST to align uniquely and perfectly to thecorresponding chromosomes with exactly the same insert size as indicatedby the eland_pair output.

Our data suggested that a large proportion of the reads mapped to chrYin the female DNA samples were false-positive signals due tonon-specific bioinformatics alignment. We reported a similar observationin our previous study using single read (SR) sequencing (Chiu et al.2008, Proc Natl. Acad. Sci. USA, 105, 20458-63). We compared if SR or PEsequencing was more prone to produce such an artifact. For PEsequencing, the reads from the two ends of each DNA fragments aregenerated independently as read1 and read2, respectively, and are pairedby post-sequencing bioinformatics. Therefore, read1 from the PEsequencing run could be analyzed as if it was SR sequencing. Whenanalyzed as SR sequencing, the absolute (and fractional) U0-1-0-0sequence reads mapped to chrY for the two female DNA samples describedabove were 147 (0.0094%) and 171 (0.0072%), respectively. This wasalmost doubled that of the corresponding accepted PE reads mapped tochrY.

As evident by the chrY data from the female samples, PE sequencing canattain higher alignment accuracy than SR sequencing. The number ofnucleotides sequenced and therefore available for alignment from eachplasma DNA molecule is doubled in PE compared with SR sequencing andthus, minimizes the chance of misalignment to other locations in thehuman genome. A positional requirement of not accepting pairs separatedby too great a distance on the same chromosome is another way ofreducing the chance of misalignment.

Identification of Trisomy 21 Fetus Using Paired-end Sequencing

Nine women each pregnant with a euploid fetus and four women eachpregnant with a T21 fetus were recruited in the first and secondtrimesters. Direct noninvasive detection of fetal T21 from maternalplasma was attempted using PE sequencing. The 13 samples were processedseparately in two PE sequencing runs. The clinical details andsequencing counts for each case are shown in Table 1 of FIG. 13.8.3-10.5 million DNA molecules were sequenced from each case, of which amedian of 1.6 million pairs (17% of total) passed the criteria to bedeemed as accepted PE reads

We expressed the number of accepted PE reads aligned to each chromosomeas a proportion of all accepted PE reads generated for the sample. Themean and SD of the proportion of accepted PE reads for chromosomes 21and X were established from the plasma samples of pregnancies with maleeuploid fetuses which were considered as the reference sample set. Thez-score, referring to the number of standard deviations from the mean ofa reference population, for each test sample was then calculated. Az-score greater than ±3 signifies a difference greater than the 99thpercentile of the proportion of accepted PE reads of the euploidreference sample set for the target chromosome, i.e., a P value of 0.01.

FIG. 14 is a bar chart of the proportion of accepted PE reads forchromosomes 21, X and Y for 13 early pregnancy maternal plasma samples.The ranges of proportion of accepted PE reads for chromosome Y for threegroups are 0.022-0.034% for the pregnancies with euploid male fetuses,0.0048-0.0058% for pregnancies with euploid female fetuses and0.029-0.038% for pregnancies with trisomy 21 male fetuses. FIG. 14 showsthat the percentages of accepted PE reads aligned to chr21 were higherfor all T21 than for euploid cases and the corresponding values for chrXwere higher and those for chrY were lower for all female than malefetuses.

Five maternal plasma samples each carrying a euploid male fetus wereselected as the reference group for the calculation of the z-scores.Z-scores were also calculated for the proportion of U0-1-0-0 reads fromread1 of the PE sequencing run to simulate the data obtained when SRsequencing was performed. Z-scores of chr21 for the four T21 fetusesranged from 5.63-8.89 for SR sequencing and ranged from 8.07-12.00 forPE sequencing. Z-scores of chrX for the four female fetuses ranged from5.04-7.69 for SR sequencing and ranged from 3.91-6.35 for PE sequencing.There were no statistically significant differences in the z-scores forchromosomes 21 or X when comparing the PE and SR sequencing data(P=0.125, Wilcoxon signed-rank test).

Size Distribution of DNA Fragments in Maternal Plasma

The length of each DNA fragment was inferred from the data output of theeland_pair program by adding 32 bp to the absolute positional offsetbetween the chromosomal positions at the start of each member of thepaired sequence reads. The awk utility of Linux was used to identify thepaired reads with a size less than or equal to each of the analyzed sizecutoffs.

We studied the size profile of plasma DNA molecules in the nine pregnantwomen carrying male fetuses among the 13 pregnancies described above aswell as plasma from 2 adult males. For the maternal plasma samples, thereads mapped to chrY are of fetal origin while the reads for the otherchromosomes are predominantly of maternal origin. We therefore analyzedthe size profile of the reads on the Y and non-Y chromosomesindependently.

FIGS. 15A and 15B are histograms showing the size distributions ofaccepted PE reads aligned to Y (black line) and non-Y (dashed line)chromosomes in (A) the plasma from an adult male and (B) the plasma froma pregnant woman carrying a male fetus, respectively. The plasma DNAsize distribution plots for the Y and non-Y chromosomes in each of theadult male samples were not statistically significantly different(P=0.118 and 0.134 respectively, Mann-Whitney rank-sum test). The plotspeaked at 167 bp and 168 bp, respectively, for the two adult malesamples, being concordant with the insert sizes of the DNA librariesobserved during bioanalyzer (Agilent) capillary electrophoresis.

For the maternal plasma samples, there was a clear demarcation betweenthe size distribution curves for the Y and non-Y chromosomes (FIG. 15B).The median (range) length of the chrY fragments for the maternal plasmasamples were 134 bp (33 bp to 378 bp) while that for the non-Ychromosomes were 157 bp (33 bp to 600 bp). The difference in sizedistribution between the Y and non-Y chromosomes for each maternalplasma sample was statistically significant (P<0.001, Mann-Whitneyrank-sum test). The size distribution of DNA fragments varies for eachchromosome.

Dependence of Fetal DNA on Size Selection

Cutoffs for DNA size were then used to achieve relative enrichment offetal DNA in maternal plasma. We compared a series of selected cutoffpoints, including 300 bp, 200 bp, 175 bp, 150 bp, 125 bp, 100 bp, 75 bpand 50 bp. The proportions of retained reads at each size cutoff areshown in FIG. 16.

FIG. 17 shows the amount of retained reads from chrY as a proportion ofall retained reads, termed retained % chrY according to an embodiment ofthe present invention. The optimal balance between the degrees of fetalDNA enrichment achieved with a reasonable retention of accepted PE readsseemed to be achieved at the cutoff points of 175 bp and 125 bp.

However, this balance is also affected by the actual detection of theoverrepresentation. Fetal chromosomal aneuploidy could be detected morereadily by maternal plasma analysis in samples with higher fractionalfetal DNA concentrations (Lo et al., 2007, Proc Natl Acad Sci USA, 104,13116-21. However, detection of overrepresentation of the aneuploidchromosome would be less precise when the absolute read number isreduced.

FIG. 18A shows the application of DNA size selection analysis for fetaltrisomy 21 detection with a plot of the z-scores for chromosome 21 ateach DNA size cutoff according to an embodiment of the presentinvention. The z-scores for chr21 for each of the size cutoffs are shownfor different samples. The known status (male euploid, female euploid,and trisomy male) of each sample is provided by a symbol described inthe legend. The horizontal dashed line refers to 3 standard divisions(SD) from the mean of the reference group.

A clearer demarcation in the z-scores of chr21 was achieved between theeuploid and T21 cases when size cutoffs of 150 bp or above were used,but at 125 bp or less, the demarcation blurred (FIG. 18A). As statedbefore, the amount of retained chr21 is also important.

FIG. 18B is a histogram showing the coefficient of variation (CV)(CV=SD/mean×100%) of measuring the proportion of retained chr21 reads ateach size cutoff using the euploid cases according to an embodiment ofthe present invention. The CV increased substantially when a size cutoffof 125 bp or less was used. Thus, FIGS. 18A and 18B confirm that amaximum length cutoff between 175 nucleotides to 125 nucleotides isoptimal, which is counterintuitive to the notion that an increasedpercentage of fetal material is always better.

In summary, the median number of unique reads for PE sequencing, namelythe accepted PE reads, was just 17% (˜1.6 million reads) of the totalsequenced reads while that for SR sequencing (U0-1-0-0 sequences ofread1) of the same sample set was 26.4% (˜2.5 million reads) of thetotal sequenced reads (FIG. 13). The difference was statisticallysignificant (P<0.001, Wilcoxon signed-rank test). The latter data weresimilar to those (23.3%, ˜2.4 million reads) reported in an earlierstudy where 28 maternal plasma samples were analyzed using SR sequencing(Chiu et al. 2008, Proc Natl. Acad. Sci. USA, 105, 20458-63).

The reduced number of unique read count for PE sequencing is possiblybecause of the more stringent definition of uniqueness whereby bothreads in a pair, i.e. 64 bp, would need to align to the reference humangenome without mismatches. Despite the reduced number of uniquesequences, PE sequencing of maternal plasma DNA allowed the detection offetal DNA and the assessment of fetal chromosome dosage, particularlywhen proper size selection was used.

We showed that one could selectively analyze the shorter sequences atthe post-sequencing stage. Selective analysis of plasma DNA sequencesshorter than a specified size cutoff would indeed increase theproportion of fetal derived sequences, but at a reduction in theabsolute number of retained sequences. The reduced total number ofsequenced reads renders the measurement of the representation of chr21less precise (FIG. 18A).

There is therefore a tradeoff between the extent of fetal DNA enrichmentand reduction in overall retained reads when any particular size cutoffvalue is used. When a maximum cutoff (i.e. length <=cutoff) is used, theoptimal value for the cutoff is between 175 and 125 nt. As mentionedabove, a minimum length may also be imposed.

The effects of the chosen size cutoff is also reflected in the CV forthe measurement of the representation of chr21. Less precisemeasurements, reflected by a larger CV (FIG. 18B), result in larger SDsand thus reduce the z-score demarcation between the aneuploid andeuploid cases. Thus, counterintuitively one cannot achieve the highestpossible diagnostic accuracy simply by simply enriching for fetal DNAsequences maximally by selecting the shortest size cutoffs, as FIG. 17would suggest.

Also, by knowing the detailed size profile of DNA molecules in maternalplasma, we could objectively predict the effects of fetal DNA enrichmentbased on size selection of the shorter sequences. The size selectionusing physical means, such as gel electrophoresis, may be performedbased on the determined size profile, and may be done in addition to thepost-sequencing selection.

In conclusion, paired-end sequencing of maternal plasma DNA permitsfetal aneuploidy detection and also provides high resolution sizeprofiles of fetal and maternal DNA in maternal plasma. Apart from usingthe Illumina Genome Analyzer (i.e. Solexa) technology for suchpaired-end sequencing, one could also use other high throughput DNAsequencing platform, e.g. the SOLiD technology from Applied Biosystems,part of Life Technologies and the Helicos True Single Molecule DNAsequencing technology (Harris TD et al. 2008 Science, 320, 106-109). Onecan also achieve the same goal by using the Roche 454 sequencingtechnology which can sequence whole DNA fragments in the targetbiological samples. Apart from trisomy 21 which has been used here as anexample of chromosomal aneuploidy, one can also apply the technologydeveloped here for the detection of aneuploidy involving chromosomes 13,18, X and Y, e.g. trisomy 13, trisomy 18 and the sex chromosomeaneuploidies.

IV. Determination of Number of Sequences Required

The sequencing result of the plasma DNA from a pregnant woman carrying aeuploid male fetus is used for this analysis. The number of sequencedtags that can be mapped without mismatches to the reference human genomesequence was 1,990,000. Subsets of sequences were randomly chosen fromthese 1,990,000 tags and the percentage of sequences aligned tochromosome 21 was calculated within each subset. The number of sequencesin the subsets was varied from 60,000 to 540,000 sequences. For eachsubset size, multiple subsets of the same number of sequenced tags werecompiled by random selection of the sequenced tags from the total pooluntil no other combination was possible. The mean percentage ofsequences aligned to chromosome 21 and its standard deviation (SD) werethen calculated from the multiple subsets within each subset size. Thesedata were compared across different subset sizes to determine the effectof subset size on the distribution of the percentage of sequencesaligned to the chromosome 21. The 5^(th) and 95^(th) percentiles of thepercentages were then calculated according to the mean and SD.

When a pregnant woman is carrying a trisomy 21 fetus, the sequenced tagsaligned to chromosome 21 should be over-represented in the maternalplasma due to an extra dose of chromosome 21 from the fetus. The degreeof over-representation is dependent on the fetal DNA percentage in thematernal plasma DNA sample following the equation below:

Per_(T21)=Per_(Eu)×(1+f/2)

wherePer_(T21) represents the percentage of sequences aligned to chromosome21 in a woman with a trisomy 21 fetus; andPer_(Eu) represents the percentage of sequences aligned to chromosome 21in a woman with a euploid fetus; andf represents the fetal DNA percentage in maternal plasma DNA

As shown in FIG. 7, the SD for the percentages of sequences aligned tochromosome 21 decreases with increasing number of sequences in eachsubset. Therefore, when the number of sequences in each subsetincreases, the interval between the 5^(th) and 95^(th) percentilesdecreases. When the 5%-95% interval for the euploid and trisomy 21 casesdo not overlap, then the differentiation between the two groups of caseswould be possible with an accuracy of >95%.

As shown in FIG. 7, the minimal subset size for the differentiation oftrisomy 21 cases from euploid cases is dependent on the fetal DNApercentage. The minimal subset sizes for differentiating trisomy 21 fromeuploid cases were 120,000, 180,000 and 540,000 sequences for fetal DNApercentages of 20%, 10% and 5%, respectively. In other words, the numberof sequences needed to be analyzed would be 120,000 for determiningwhether a fetus has trisomy 21 when a maternal plasma DNA samplecontains 20% fetal DNA. The number of sequences needed to be analyzedwould be increased to 540,000 when the fetal DNA percentage drops to 5%.

As the data were generated using 36 basepair sequencing, 120,000,180,000 and 540,000 sequences correspond to 0.14%, 0.22% and 0.65% ofthe human genome, respectively. As the lower range of fetal DNAconcentrations in maternal plasma obtained from early pregnancies werereported to be some 5% (Lo, YMD et al. 1998 Am J Hum Genet 62, 768-775),the sequencing of about 0.6% of the human genome may represent theminimal amount of sequencing required for diagnosis with at least 95%accuracy in detecting fetal chromosomal aneuploidy for any pregnancy.

V. Random Sequencing

To illustrate that the sequenced DNA fragments were randomly selectedduring the sequencing run, we obtained the sequenced tags generated fromthe eight maternal plasma samples analyzed in example I. For eachmaternal plasma specimen, we determined the starting positions inrelation to the reference human genome sequence, NCBI assembly 36, ofeach of the 36 bp sequenced tags that were aligned uniquely tochromosome 21 without mismatches. We then ordered the starting positionnumber for the pools of aligned sequenced tags from each specimen inascending order. We performed a similar analysis for chromosome 22. Forillustrative purpose, the top ten starting positions for chromosome 21and chromosome 22 for each of the maternal plasma specimens are shown inFIGS. 8A and 8B, respectively. As can be appreciated from these Tables,the sequenced pools of DNA fragments were non-identical between samples.

Any of the software components or functions described in thisapplication, may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a computer readable medium for storage and/ortransmission, suitable media include random access memory (RAM), a readonly memory (ROM), a magnetic medium such as a hard-drive or a floppydisk, or an optical medium such as a compact disk (CD) or DVD (digitalversatile disk), flash memory, and the like. The computer readablemedium may be any combination of such storage or transmission devices.

Such programs may also be encoded and transmitted using carrier signalsadapted for transmission via wired, optical, and/or wireless networksconforming to a variety of protocols, including the Internet. As such, acomputer readable medium according to an embodiment of the presentinvention may be created using a data signal encoded with such programs.Computer readable media encoded with the program code may be packagedwith a compatible device or provided separately from other devices(e.g., via Internet download). Any such computer readable medium mayreside on or within a single computer program product (e.g. a hard driveor an entire computer system), and may be present on or within differentcomputer program products within a system or network. A computer systemmay include a monitor, printer, or other suitable display for providingany of the results mentioned herein to a user.

An example of a computer system is shown in FIG. 9. The subsystems shownin FIG. 9 are interconnected via a system bus 975. Additional subsystemssuch as a printer 974, keyboard 978, fixed disk 979, monitor 976, whichis coupled to display adapter 982, and others are shown. Peripherals andinput/output (I/O) devices, which couple to I/O controller 971, can beconnected to the computer system by any number of means known in theart, such as serial port 977. For example, serial port 977 or externalinterface 981 can be used to connect the computer apparatus to a widearea network such as the Internet, a mouse input device, or a scanner.The interconnection via system bus allows the central processor 973 tocommunicate with each subsystem and to control the execution ofinstructions from system memory 972 or the fixed disk 979, as well asthe exchange of information between subsystems. The system memory 972and/or the fixed disk 979 may embody a computer readable medium.

The above description of exemplary embodiments of the invention has beenpresented for the purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdescribed, and many modifications and variations are possible in lightof the teaching above. The embodiments were chosen and described inorder to best explain the principles of the invention and its practicalapplications to thereby enable others skilled in the art to best utilizethe invention in various embodiments and with various modifications asare suited to the particular use contemplated.

The present application is related to concurrently filed non-provisionalapplication entitled “DETERMINING A NUCLEIC ACID SEQUENCE IMBALANCE,”(Attorney Docket No. 016285-005210US) the entire contents of which areherein incorporated by reference for all purposes.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

1. A method for performing prenatal diagnosis of a fetal chromosomalaneuploidy in a biological sample obtained from a pregnant femalesubject, wherein the biological sample includes nucleic acid molecules,the method comprising: receiving the biological sample; sequencing atleast a portion of a plurality of the nucleic acid molecules containedin the biological sample, wherein the sequenced portion represents afraction of the human genome; based on the sequencing: determining afirst amount of a first chromosome from sequences identified asoriginating from the first chromosome; determining a second amount ofone or more second chromosomes from sequences identified as originatingfrom one of the second chromosomes; determining a parameter from thefirst amount and the second amount; comparing the parameter to one ormore cutoff values; and based on the comparison, determining aclassification of whether a fetal chromosomal aneuploidy exists for thefirst chromosome.
 2. The method of claim 1, wherein the sequencing isperformed randomly on a portion of the nucleic acid molecules containedin the biological sample.
 3. The method of claim 1, wherein thebiological sample is maternal blood, plasma, serum, urine or saliva 4.The method of claim 1, wherein the biological sample is transcervicallavage fluid.
 5. The method of claim 1, wherein the first chromosome ischromosome 21, chromosome 18, chromosome 13, chromosome X, or chromosomeY.
 6. The method of claim 1, wherein the parameter is a ratio ofsequences that originate from the first chromosome.
 7. The method ofclaim 6, wherein the ratio is obtained from any one or more of afractional count of the number of sequenced tags, a fractional number ofsequenced nucleotides, and a fractional length of accumulated sequences.8. The method of claim 6, wherein the sequences that originate from thefirst chromosome are selected to be less than a specified number of basepairs.
 9. The method of claim 8, wherein the specified number of basepairs is 300 bp, 200 bp, or 100 bp.
 10. The method of claim 1, whereinthe nucleic acid molecules of the biological sample have been enrichedfor sequences originating from at least one particular chromosome. 11.The method of claim 1, wherein the nucleic acid molecules of thebiological sample have been enriched for sequences less than 300 bp. 12.The method of claim 1, wherein the nucleic acid molecules of thebiological sample have been enriched for sequences less than 200 bp. 13.The method of claim 1, wherein the nucleic acid molecules of thebiological sample have been amplified using a polymerase chain reaction.14. The method of claim 1, wherein the sequenced portion represents atleast a pre-determined fraction of the human genome.
 15. The method ofclaim 1, wherein the fraction represents at least 0.1% of the humangenome.
 16. The method of claim 1, wherein the fraction represents atleast 0.5% of the human genome.
 17. The method of claim 1, wherein atleast one of the cutoff values is related to the fractionalconcentration of fetal DNA in the biological sample.
 18. The method ofclaim 17, wherein the fractional concentration of fetal DNA in thebiological sample is determined by any one or more of a proportion of Ychromosome sequences, a fetal epigenetic marker, or using singlenucleotide polymorphism analysis.
 19. The method of claim 1, wherein acutoff value is a reference value established in a normal biologicalsample.
 20. The method of claim 1, further comprising: identifying anamount of fetal DNA in the biological sample; and calculating a number Nof sequences to be analyzed based on a desired accuracy.
 21. A computerprogram product comprising a computer readable medium encoded with aplurality of instructions for controlling a computing system to performan operation for performing prenatal diagnosis of a fetal chromosomalaneuploidy in a biological sample obtained from a pregnant femalesubject, wherein the biological sample includes nucleic acid molecules,the operation comprising the steps of: receiving data from a randomsequencing of a portion of the nucleic acid molecules contained in thebiological sample obtained from a pregnant female subject, wherein thebiological sample includes nucleic acid molecules, wherein the portionrepresents a fraction of the human genome; based on the data from therandom sequencing: determining a first amount of a first chromosome fromsequences identified as originating from the first chromosome;determining a second amount of one or more second chromosomes fromsequences identified as originating from one of the second chromosomes;determining a parameter from the first amount and the second amount;comparing the parameter to one or more cutoff values; and based on thecomparison, determining a classification of whether a fetal chromosomalaneuploidy exists for the first chromosome.
 22. A method for performingprenatal diagnosis of a fetal chromosomal aneuploidy in a biologicalsample obtained from a pregnant female subject, wherein the biologicalsample includes nucleic acid molecules, the method comprising: receivingthe biological sample; calculating a number N of sequences to beanalyzed based on a desired accuracy; randomly sequencing at least N ofthe nucleic acid molecules contained in the biological sample, whereinthe portion represents a fraction of the human genome; based on therandom sequencing: determining a first amount of a first chromosome fromsequences identified as originating from the first chromosome;determining a second amount of one or more second chromosomes fromsequences identified as originating from one of the second chromosomes;determining a parameter from the first amount and the second amount;comparing the parameter to one or more cutoff values; and based on thecomparison, determining a classification of whether a fetal chromosomalaneuploidy exists for the first chromosome.
 23. The method of claim 22,further comprising: identifying a percentage of fetal DNA in thebiological sample, wherein calculating a number N of sequences to beanalyzed based on a desired accuracy is based on the percentage.